Project Abstract This is an application for renewal of a grant to study picornavirus genome replication. Studies of poliovirus (PV) continue to establish paradigms for the molecular and cellular biology of all positive-strand RNA viruses capable of causing morbidity and/or mortality in humans. PV replicates its genome in association with membranes. In fact, the virus creates its own genome-replication organelle (RO) with a unique lipid composition, including an abundance of the phosphoinositide (PIP), phosphatidylinositol-4-phosphate (PI4P). During the past five years, many laboratories have been in search of the mechanism by which PI4P biosynthesis is induced by various picornaviruses, including PV. In general, these studies tested the hypothesis that a single viral protein hijacks a single cellular PI4 kinase (PI4K), leading to kinase relocalization and synthesis of PI4P. Because of the long-established connection between the enteroviral 3A(B) protein and the guanine nucleotide exchange factor, GBF1, most of the early studies focused on 3A(B) and concluded that this viral protein is responsible for hijacking a PI4K, often by an indirect mechanism. However, this once-held consensus opinion has now returned to uncertainty. Many years ago, our laboratory obtained genetic evidence of a possible role of 3CD in the biogenesis of PV RO. During the previous funding period, we made a definitive connection between 3CD and RO biogenesis by showing that 3CD is both necessary and sufficient for induction of PI4P biosynthesis in cells. We demonstrated that the normal cellular GBF1-Arf1-PI4K axis is employed. We identified two derivatives of 3CD with amino acid substitutions in the 3C domain (3CmD) or 3D domain (3CDm) that are defective for induction of PI4P biosynthesis at discrete steps in this pathway. In both instances, the derivatives exhibit perturbations to PIP-binding activity of 3CD. In addition to PI4P, 3CD also induces PI(4,5)P2 (PIP2) biosynthesis in cells. PIP2 induction does not arise from the 3CD-dependent increase in PI4P but appears to be a distinct process based on the observation that both 3CmD and 3CDm proteins remain competent for PIP2 induction. PV 3CD is a PIP-binding protein and a regulator of multiple PIP biosynthetic pathways. Our proposed studies aim to address how and why. During the next funding period, we will pursue the following specific aims: (1) Define the structure-function relationships of the PIP-binding domains of 3C and 3D alone and in the context of 3CD; (2) Elucidate the mechanism of induction of PI4P biosynthesis by 3CD alone and in the context of infection; and (3) Elucidate the mechanism of induction of PIP2 biosynthesis by 3CD alone and in the context of infection.